Use of pentagastrin to inhibit gastric acid secretion or as a diuretic

ABSTRACT

This invention pertains to the discovery that pentagastrin, when administered in conjunction with a proton pump inhibitor (PPI) is synergistic with the PPI and significantly increases the efficacy of the PPI in reducing/mitigating excess gastric acid secretion.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. Ser. No. 11/608,667, filed onDec. 8, 2006, which is a continuation of U.S. Ser. No. 09/671,764, filedon Sep. 27, 2000, which claims priority to and benefit of U.S. Ser. No.60/156,491, filed on Sep. 28, 1999, all of which are incorporated hereinby reference in their entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under a grant awarded bythe Veterans Administration. The Government of the United States ofAmerica may have certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to the treatment of physiological disorderscharacterized by excess gastric acid secretion. In particular, thisinvention relates to the use of pentagastrin as a synergist with protonpump inhibitors (PPIs).

BACKGROUND OF THE INVENTION

A wide number of pathological conditions are characterized byoversecretion of gastric acid. Such conditions include, but are notlimited to, Zollinger/Ellison syndrome (ZES), gastroesophageal refluxdisease, peptic ulcer disease, duodenal ulcers, atrophic gastritis,esophagitis, and the like. Conditions such as ZES and peptic ulcers, inparticular, can have serious complications and represent some of themost prevalent diseases in industrialized nations.

The treatment of such conditions often requires high and repeated dosesof acid output (AO) inhibiting agents to effectively reduce intragastricacidity. Although histamine H₂-antagonists have been used successfullyto treat such conditions, the erratic and diminishing responses withthese antagonists as well as the progressive occurrence of more severside effects associated with the use of larger doses has led to the useof the more effective proton pump inhibitors (PPIs).

Proton pump inhibitors (PPI) are potent inhibitors of gastric acidsecretion by inhibiting H⁺/K⁺-ATPase, the enzyme involved in the finalstep of hydrogen ion production in the parietal cells. Hence, PPI havebeen used in the treatment of gastric acid related diseases in humans.Despite their wide-spread use, means of increasing PPI efficacy, e.g. ata lower dose are desired.

SUMMARY OF THE INVENTION

This invention provides a novel method of treating pathologicalconditions characterized by excess gastric acid secretion. Inparticular, this invention pertains to the discovery that administrationof pentagastrin (an agent that is typically used to increase acidsecretion), in conjunction with a proton pump inhibitor (PPI) willresult in increased efficacy (e.g. prolonged effect and/or greatereffect at reduced dosage) than use of the proton pump inhibitor alone.The effect is also mediated by gastrin and gastrin or pentagastrinanalogues or derivatives. In particular embodiments, thepentagastrin/PPI combination appears synergistic.

Thus, in one embodiment, this invention provides methods of increasingthe efficacy of a gastric H⁺/K⁺-ATPase pump inhibitor (PPI) in a mammal(e.g. a rodent, largomorph, bovine, canine, equine, non-human primate,human, etc.). The methods preferably involve administering to the mammalpentagastrin, gastrin or analogues or derivatives thereof in conjunctionwith a gastric proton pump inhibitor. Pentagastrin is used inparticularly preferred embodiments. The pentagastrin can be administeredbefore, simultaneously with, or after the PPI, but in a most preferredembodiment, the pentagastrin administration precedes the PPIadministration. In addition to the use of exogenous gastrin orpentagastrin, the method can involve upregulating endogenous gastrinsecretion using, for example, aromatic amino acids, or with a meal, etc.Essentially anything that stimulates G-cell activity will increase theefficacy of a PPI.

Administration of the gastrin/pentagastrin/analogue and the PPI can beby any route convenient for the application of these agents. Inpreferred embodiments, the gastrin/pentagastrin/analogue is administeredby injection (e.g. subcutaneous injection) and the PPI is administeredorally or by injection (e.g. intravenous injection). Particularlypreferred pentagastrin/gastrin/analogue dosages range from about 0.1mg/kg/hr to about 10 mg/kg/hr.

The mammal is preferably a mammal diagnosed with a pathologycharacterized by excess gastric acid secretion, e.g., Zollinger/Ellisonsyndrome (ZES), gastroesophageal reflux disease (GERD), peptic ulcerdisease, atrophic gastritis, esophagitis, stress induced hypersecretion,and/or idiopathic gastric acid hypersecretion. Preferred proton pumpinhibitors used in this invention include, but are not limited torabeprazole, omeprazole, lansoprazole, and pantoprazole, as well ascogeners or racemic mixtures of the same. The mammal may also sufferfrom hypersensivity to normal acid secretion that may result ingastrointestinal inflammation and ulceration.

This invention also involves the discovery that administration ofpentagastrin gastrin or analogues thereof or other compounds that act atthe same receptor site (e.g. that are agonistic at the cholecystokinin(CCK) receptor (see, U.S. Pat. No. 5,319,073 for a description of theCCK receptor) will increase urinary sodium excretion (e.g. gastrin,pentagastrin, cholecystokinin and derivatives or analogues thereof actas a diuretic). Thus, in another embodiment, this invention providesmethods of increasing urinary sodium excretion and free water excretion.These methods involve administering to a mammal diagnosed with apathological condition characterized by excessive fluid retention, adose of pentagastrin or analogues thereof (or in certain embodiments,gastrin or analogues thereof) sufficient to increase urinary sodiumexcretion in the mammal. In preferred embodiments, the pathologicalcondition is high blood pressure, fluid retention associated with heartfailure, fluid retention associated with acute or chronic kidneyfailure, fluid retention associated with cirrhosis, calcium kidneystones, nephrogenic diabetes insipidus, renal tubular acidosis,treatment of Meniere's disease, constrictive pericarditis, andhepatorenal syndrome. The pentagastrin is typically administered in thedosage ranges indicated above.

Kits are also provided for the practice of the methods of thisinvention. A preferred kit for the treatment of a pathologycharacterized by excess gastric acid secretion, said kit comprises acontainer containing a proton pump inhibitor (PPI); and a containercontaining pentagastrin. Preferred proton pump inhibitors include, butare not limited to rabeprazole, omeprazole, lansoprazole, andpantoprazole, as well as cogeners or racemic mixtures of the same. Thepentagastrin and/or the PPI can be provided in a pharmaceuticallyacceptable excipient or diluent. The kits can additionally includematerials describing the use of pentagastrin, gastrin or analoguesthereof in conjunction with a PPI to reduce gastric acid secretionand/or materials describing the use of pentagastrin as a diuretic.Instructional materials can also include recommended dosages anddescription(s) of counterindications, etc.

Also included are kits for increasing urinary sodium excretion in amammal. Preferred kits comprise a container containing a pentagastrin,gastrin, or analogue thereof; and instructional materials describing theuse of said pentagastrin, gastrin, or analogue thereof to increaseurinary sodium excretion in a mammal.

DEFINITIONS

The “H⁺/K⁺-ATPase” or “proton pump” is an acid pump responsible for thefinal step of acid secretion (see, e.g., Besancon et al. (1996) J. Biol.Chem. 272: 22438-22446; Lambrecht et al. (1998) J. Biol. Chem.273:13719-28; Nwokolo et al. (1991) Gut; 32:1455; Sachs et al.: (1995)Ann Rev Pharm Toxic. 35: 277-305. It is believed the “proton pump” is aheterodimer. The catalytic subunit has ten membrane-inserted segmentsand the beta subunit a single transmembrane segment. The proton pumptypically catalyzes an electroneutral H⁺ for K⁺ exchange.

“Proton pump inhibitors (PPIs)” are compounds that are inhibit activityof the, H+/K+-adenosine triphosphatase (ATPase) proton pump. A largernumber of proton pump inhibitors are well known to those of skill in theart. Certain preferred PPIs include substituted pyridyl methylsulfinylbenzimidazoles. These compounds accumulate in the acid space of theparietal cell and convert to active sulfonamide by an acid-catalyzedreaction. Consequent covalent inhibition of H⁺/K⁺-ATPase blocks thefinal step of acid secretion. PPIs include, but are not limited torabeprazole, omeprazole (Prilosec™, Antra™, Audazol™, Desec™,Gastroloc™, Losec™, Miracid™, Mopral™, Zefxon™), lansoprazole((Prevacid™), and pantoprazole.

The phrase “in conjunction with” when used in reference to the use ofproton pump inhibitors in conjunction with pentagastrin indicates thatthe PPI and the pentagastrin are administered so that there is at leastsome chronological overlap in their physiological activity on theorganism. Thus the PPI and pentagastrin can be administeredsimultaneously and/or sequentially. In sequential administration theremay even be some substantial delay (e.g., minutes or even hours or days)before administration of the second agent as long as the firstadministered agent has exerted some physiological alteration on theorganism when the second administered agent is administered or becomesactive in the organism.

The term mammal includes essentially any mammal including, but notlimited to dogs, cats, sheep, cattle, horses, goats, mice, rabbits,hamsters, pigs, monkeys and other non-human primates, and humans. Thus,veterinary as well as medical applications of this invention arecontemplated.

The term “conservative substitution” is used in reference to proteins orpeptides to reflect amino acid substitutions that do not substantiallyalter the activity (specificity or binding affinity) of the molecule.Typically conservative amino acid substitutions involve substitution oneamino acid for another amino acid with similar chemical properties (e.g.charge or hydrophobicity). The following six groups each contain aminoacids that are typical conservative substitutions for one another: 1)Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

The phrase “pentagastrin/gastrin/analogue” refers to a pentagastrin, agastrin, or an analogue or derivative of a gastrin or a pentagastrin,e.g. as described herein.

The phrase “treatment of a pathology” refers to the amelioration ormitigation or elimination of one or more symptoms of the pathology.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Calculation of onset and duration. The first arrow indicates thetime point when AO falls to <10 mEq/h and the average rate over the next1 h remains below this value. The second arrow indicates the time pointat which AO is >10-mEq/h; the time between arrows is the duration ofaction value. The time of onset and time span of the longest durationwhen AO remained >10 mEq/h over the 24-h period was used in thecalculation of the mean values. If subjects' AO never fell to <10 mEq/h,the onset was set as missing and duration was set as 0, so as not tobias the mean. Collections were made every 15 min during the first 2 hof the study, then every 30 min after that.

FIG. 2. Mean cumulative AO over the 25-h period after a single i.v. doseof pantoprazole (20, 40, 80, and 120 mg), i.v. famotidine (20 mg), orplacebo. Note that AO remained constant for the placebo group over theentire 25-h period of PG administration.

FIG. 3. Mean rate of AO over 24 h for IV pantoprazole (20, 40, 80, or120 mg), i.v. famotidine (20 mg), or placebo.

FIG. 4A shows meantime of onset and FIG. 4B shows duration of responsefor escalating doses of i.v. pantoprazole or a single i.v. dose offamotidine. Onset of response is defined as time until AO decreased to<10 mEq/h and remained below this level for 1 h. Duration of response isdefined as time from onset until time when AO levels rose to >10 mEq/hand remained so for 1 h. Values are mean ±SD.

FIG. 5A through FIG. 5E show the effects of meal gavage on renalhemodynamics, sodium excretion and urine volume in rats. Following abasal interval, the rats were infused with either L-365,260 (80pmoles/kg, 5 min bolus) (closed circles) or vehicle alone (open circles)prior to meal gavage with standard rat chow (2.5 ml/hr) and thefollowing parameters of renal function were measured: FIG. 7A showsdoppler renal artery blood flow (RBF); FIG. 5B shows glomerularfiltration rate (GFR); FIG. 5C shows urinary sodium excretion, (UNaV);FIG. 5D shows fractional sodium excretion (FeNa);

FIG. 5E shows urinary output, (UV). Each time period shown represents 20minutes. Within-group comparisons (*, compared to basal, P<0.05) (n=5)were made using analysis of variance (ANOVA), Tukey's test.Between-group comparisons (#) were made using the t-test (#, P<0.05)(n=5). No effect on Mean Arterial Pressure (MAP) was observed during thestudy period (data not shown).

FIG. 6A through 6E shows the effects of intrarenal administration ofgastrin on renal hemodynamics, renal sodium excretion and urine volumesin rats. Gastrin (10 pmoles/kg/hr) with or without the CCKBR antagonist,L-365,260 (80 pmoles/kg, 5 min bolus) was infused via the right renalartery and the following parameters were measured:

FIG. 6A shows doppler renal artery blood flow (RBF); FIG. 6B showsglomerular filtration rate (GFR); FIG. 6C shows urinary sodiumexcretion, (UNaV); FIG. 6D shows fractional sodium excretion (FeNa);FIG. 6E shows urinary output, UV. Each time period shown represents 20minutes. Within-group comparisons were made using analysis of variance(ANOVA), Tukey's test (*, compared to basal; #, L-365,260 versusvehicle: P<0.05) (n=5). No effect on Mean Arterial Pressure (MAP) wasobserved during the study period (data not shown).

FIG. 7 shows the effect of meal stimulated gastrin on Na⁺-K⁺-ATPaseactivity in the rat proximal tubule and medullary thick ascending limbof Henle. L-365,260 (80 pmoles/kg, 5 min bolus, filled bars, n=6) orvehicle alone (open bars, n=5) was infused into the right renal arteryprior to gastrin stimulation by meal gavage with standard rat chow (2.5ml/hr). Na⁺-K⁺-ATPase activity was measured separately in the proximaltubule and medullary thick ascending limb of Henle following the meal.Data are expressed as the mean ±SD of the percent inhibition inNa⁺-K⁺-ATPase activity measured in the absence of a meal and withinfusion of vehicle alone (n=3).

DETAILED DESCRIPTION

A wide number of pathological conditions are characterized byoversecretion of gastric acid. Such conditions include, but are notlimited to Zollinger/Ellison syndrome (ZES), gastroesophageal refluxdisease, peptic ulcer disease, duodenal ulcers, atrophic gastritis,esophagitis. In particular, conditions such as ZES and peptic ulcers canhave serious complications.

Peptic ulcers are one of the most prevalent diseases in industrializednations. Control of gastric acid secretion is the main therapy forpeptic ulcers. Gastric acid secretion is, in turn, brought about by theinteraction of three physiological stimulants, gastrin, acetylcholineand histamine with their respective parietal cell receptors. Prior tothe discovery of histamine H₂-receptor antagonists such as cimetidineand ranitidine, peptic ulcer treatment consisted of antacid therapy andanticholinergic drugs (e.g. dicyclomine HCl). With the advent ofH₂-receptor antagonists, however, treatment with anticholinergic agentshas been largely supplanted by histamine H₂-receptor antagonist therapy.The development of this class of therapeutic entities presents one ofthe most important advances in the field of medicinal chemistry.

Another major development in the treatment of peptic ulcers has beenrealized with the introduction of H⁺/K⁺-ATPase inhibitors e.g.,omeprazole. The enzyme H⁺/K⁺-ATPase, which is also known as the protonpump, is located in the membrane of gastric parietal cells and isresponsible for the transport of protons from blood to lumen, which, inturn, results in decreasing the pH of stomach contents which leads toaggravation of peptic ulcers.

This invention pertains to the discovery that administration ofpentagastrin (an agent that is typically used to increase acidsecretion) in conjunction with a proton pump inhibitor (PPI) will resultin increased efficacy (e.g. prolonged effect and/or greater effect atreduced dosage) than use of the proton pump inhibitor alone. Inparticular embodiments, the pentagastrin/PPI combination appearssynergistic.

Thus, in one embodiment, this invention provides methods of increasingthe efficacy of a gastric H⁺/K⁺-ATPase pump inhibitor (PPI) in a mammal(e.g. a rodent, largomorph, bovine, canine, equine, non-human primate,human, etc.). The methods preferably involve administering to the mammalpentagastrin in conjunction with the gastric proton pump inhibitor. Thepentagastrin can be administered before, simultaneously with, or afterthe PPI, but in a most preferred embodiment, the pentagastrinadministration precedes the PPI administration.

It was also a discovery of this invention that essentially any increasein gastrin level in conjunction with a PPI will result in increasedefficacy of the PPI. Thus, instead of pentagastrin administration,exogenous gastrin can be supplied. Alternatively endogenous gastrinsecretion can be upregulated using, for example, aromatic amino acids,or with a meal, etc. Thus, it is believed that essentially anything thatstimulates G-cell activity will increase the efficacy of a PPI.

Thus, in various embodiments, this invention contemplates administrationof a PPI or combination of PPIs in conjunction with pentagastrin, and/orgastrin, and/or pentagastrin analogues, and/or gastrin analogues toincrease the efficacy of the PPI(s).

Proton Pump Inhibitors.

Proton pump inhibitors (PPIs) are compounds that are selectively inhibitactivity of the gastric acid pump, H⁺/K⁺-adenosine triphosphatase(ATPase). Preferred PPIs include substituted pyridyl methylsulfinylbenzimidazoles. These compounds accumulate in the acid space of theparietal cell and convert to active sulfonamide by an acid-catalyzedreaction. Consequent covalent inhibition of H⁺/K⁺-ATPase blocks thefinal step of acid secretion. Other preferred PPIs include varioussubstituted benzimidazoles. Commercially available PPIs include, but arenot limited to, omeprazole, lansoprazole, and pantoprazole.

Numerous proton pump inhibitors are known to those of skill. Thus, forexample, U.S. Pat. No. 6,093,738 describes novel thiadiazole compoundsthat are effective as proton pumps inhibitors. European Patent Nos.322133 and 404322 disclose quinazoline derivatives, European Patent No.259174 describes quinoline derivatives, and WO 91/13337 and U.S. Pat.No. 5,750,531 offer pyrimidine derivatives, as proton pump inhibitors.

Suitable proton pump inhibitors are also disclosed, for example inEP-A1-0005129, EP-A1-174 726, EP-A1-166 287, GB 2 163 747 andWO90/06925, WO91/19711, WO91/19712, WO94/27988 and WO95/01977.

Particularly preferred PPIs include, but are not limited to omeprazole,lansoprazole, and pantoprazole and derivatives or analogues thereof. Onesuch derivative is s-omeprazole (Nexium™).

The proton pump inhibitors used in the dosage forms of the invention canbe used in neutral form or in the form of a salt (e.g., an alkalinesalt), such as for instance the Mg²⁺, Ca²⁺, Na⁺, K⁺, or Li⁺ salts,preferably the Mg²⁺ salts. Further where applicable, the compounds canbe used in racemic form or in the form of a substantially pureenantiomer thereof, or salts of the racemates or the single enantiomers.

In addition this invention contemplates the use of a single proton pumpinhibitor, or in certain embodiments, combinations of two or more protonpump inhibitors.

Proton pump inhibitors are commercially available. In addition,synthesis protocols are well known to those of skill in the art (see,e.g., European Patent Nos. 322133, 404322, 259174, EP-A1-0005129,EP-A1-174 726, EP-A1-166 287, PCT Patent Applications WO 91/13337,WO90/06925, WO91/19711, WO91/19712, WO94/27988 and WO95/01977, U.S. Pat.No. 5,750,531, etc.)

Pentagastrin.

Pentagastrin(N-t-butyloxycarbonyl-Beta-alanyl-L-tryptophyl-L-methionyl-L-aspartyl-L-phenyl-alanylamide, SEQ ID NO:1) is a pentapeptide containing a gastrin carboxylterminal tetrapeptide, the active portion found in essentially allnatural gastrins. Pentagastrin is a colorless crystalline solid solublein dimethylformamide and dimethylsulfoxide; it is almost insoluble inwater, ethanol, ether, benzene, chloroform, and ethyl acetate.Pentagastrin contains the C-terminal tetrapeptide responsible for theactions of the natural gastrins and, therefore, acts as a physiologicgastric acid secretagogue. The recommended dose of 6 μg/kgsubcutaneously (in applications where increased gastric acid secretionis desired) produces a peak acid output which is reproducible when usedin the same individual. Pentagastrin stimulates gastric acid secretionapproximately ten minutes after subcutaneous injection, with peakresponses occurring in most cases twenty to thirty minutes afteradministration. Pentagastrin is typically used as a diagnostic agent forevaluation of gastric acid secretory function. In one preferredformulation, pentagastrin is formulated with sodium chloride and waterfor injection. The pH is typically adjusted with ammonium hydroxide andor hydrochloric acid. In one commercially available formulation, each mlof injection contains 0.25 mg (250 mcg) pentagastrin along with 8.8 mgsodium chloride and water for injection, USP.

The methods of this invention are not limited to the use ofpentagastrin. To the contrary, it was a discovery of this invention thatin addition to pentagastrin, exogenous gastrin can be supplied orendogenous gastrin secretion can be upregulated using, for example,aromatic amino acids, or with a meal, etc. Thus, it is believed thatessentially anything that stimulates G-cell activity will increase theefficacy of a PPI.

Thus, in addition to gastrin and pentagastrin, this inventioncontemplates the use of gastrin or pentagastrin analogues orderivatives. Such analogues or derivatives are well known to those ofskill in the art. Such variants include, but are not limited to the 34-,17-, and 14-amino acid species of gastrin, and other truncation variantscomprising the active C-terminal tetrapeptide (TrpMetAspPhe-NH₂, (SEQ IDNO: 2) which is reported in the literature to have full pharmacologicalactivity (see Tracey and Gregory (1964) Nature (London), 204: 935). Alsoincluded are variants of gastrin and/or truncated gastrins where nativeamino acids are replaces with conservative substitutions. Also includeare various analogues of these molecules, including, but not limited tothe N-protected derivative Boc-TrpMetAspPhe-NH2, SEQ ID NO:3).

In addition, it is noted that gastrins are structurally related to theCCK's are structurally-related neuropeptides which exist ingastrointestinal tissue and in the CNS (see Mutt V., GastrointestinalHormones, Glass G. B. J., ed., Raven Press, N.Y., p 169 and Nisson G.,ibid, 127). Thus it is believed that CCKs or analogues or derivativesthereof that stimulate endogenous gastrin secretion or that generallystimulate G-cell activity will be useful in the methods of thisinvention.

Gastrins, pentagastrins, or analaogues are commercially available. Inaddition synthetic protocols are well known. Thus, for example,pentagastrin can be chemically synthesized using well known peptidesynthesis methodologies (see, e.g. Barany and Merrifield Solid-PhasePeptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis,Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A.;Merrifield et al. (1963) J. Am. Chem. Soc., 85: 2149-2156; and Stewartet al. (1984) Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co.,Rockford, Ill.).

Combining PPI and Pentagastrin and, Optionally, Antibiotics.

The proton pump inhibitor (PPI) and the pentagastrin (or gastrin oranalogue etc.) can be administered simultaneously. However, in apreferred embodiment, the pentagastrin or analogue thereof isadministered first followed by the PPI. In certain embodiments, thepentagastrin or analogue thereof can be administered after the PPI.

The methods of this invention are not limited to the use of a singlepentagastrin/analogue or to the use of a single PPI. In certainembodiments, combinations of two or more PPIs and/or two or morepentagastrin/analogues are contemplated.

In certain embodiments, it is desirable to administer one or moreantibiotics in conjunction with the PPI and pentagastrin. Thus, forexample, the treatment of ulcers associated with Helicobacter spinfection (e.g. Helicobacter pylori), the antibiotic willmitigate/eliminate the bacterial component of the pathology.

It is noted that U.S. Pat. No. 5,629,305 teaches that a proton pumpinhibitor (e.g. omeprazole or lansoprazole) which increases intragastricpH, can increase the bioavailability of various antibiotics, inparticular the therapeutic amount of an acid degradable antibacterialcompound such as a penicillin or a macrolide. A wide variety ofantibiotics are suitable for use with the methods of this invention.Such antibiotics include, but are not limited to penicillin basedantibiotics, tetracyclines, macrolides, cephalosporins,fluoroguinolones, and the like.

Pharmaceutical Formulations and Administration Thereof.

The gastrin and/or pentagastrin or derivatives or analogues thereof,and/or PPI(s) used in the methods of this invention, (e.g. thetherapeutic catalytic antagonists) are preferably administered byintravenous, parenteral, or oral means. The active molecules (e.g., PPIor pentagastrin) are typically combined with a pharmaceuticallyacceptable carrier (excipient) to form a pharmacological composition.Pharmaceutically acceptable carriers can contain a physiologicallyacceptable compound that acts, for example, to stabilize the compositionor to increase or decrease the absorption of the agent. Physiologicallyacceptable compounds can include, for example, carbohydrates, such asglucose, sucrose, or dextrans, antioxidants, such as ascorbic acid orglutathione, chelating agents, low molecular weight proteins,compositions that reduce the clearance or hydrolysis of the anti-mitoticagents, or excipients or other stabilizers and/or buffers.

Other physiologically acceptable compounds include wetting agents,emulsifying agents, dispersing agents or preservatives which areparticularly useful for preventing the growth or action ofmicroorganisms. Various preservatives are well known and include, forexample, phenol and ascorbic acid. One skilled in the art wouldappreciate that the choice of a pharmaceutically acceptable carrier,including a physiologically acceptable compound depends, for example, onthe route of administration of the PPI/pentagastrin and on theparticular physio-chemical characteristics of the agent.

In various embodiments the gastrin/pentagastrin/analogue and/or the PPIcan be provided in a substantially dry and/or pure form to be combinedwith a diluent/excipient at the time of use or one or both agents can beprovided already combined with an appropriate excipient (e.g. in a unitdosage form). In certain embodiments, the gastrin/pentagastrin/analogureor the PPI is provided in a dry (e.g. lyophilized/dehydrated) form,while the other component is suspended in a fluid excipient. Addition ofthe dry component to the excipient results in the admixture of thegastrin/pentagastrin/analogure and the PPI. In other embodiments, boththe pentagastrin and the PPI are provided combined in a compatibleexcipient.

It is noted that, in certain embodiments, the PPI and thegastrin/pentagastrin/analogue can be provided combined with differentexcipients but in a single unit dosage form. Thus, for example, a tabletcan comprise two lamina, one lamina containing the pentagastrin and afirst excipient and the second lamina containing the PPI and a secondexcipient. If necessary the lamina can be separated by an inert/neutrallayer. Other “multi-excipient” systems can be similarly formulated (e.g.as time release particles in a capsule, dual container gelatin capsules,etc.).

Thus, the pharmaceutical compositions can be administered in a varietyof unit dosage forms depending upon the method of administration. Forexample, unit dosage forms suitable for oral administration includepowder, tablets, pills, capsules and lozenges. Certain therapeuticmolecules of this invention may be only marginally soluble in aqueoussolutions. In a preferred embodiment, these compositions are typicallysolubilized, emulsified or suspended in an acceptable excipient.

It is noted that pharmaceutically acceptable formulations forpentagastrin and PPIs are well known to those of skill in the art. Thus,the PPI and the pentagastrin can be administered in the formulation(s)and by the means typically used for these drugs. In particularlypreferred embodiments, the pentagastrin is administered by subcutaneousinjection.

The concentration of therapeutic agent in these formulations can varywidely, and will be selected primarily based on fluid volumes,viscosities, body weight and the like in accordance with the particularmode of administration selected and the patient's needs. Actual methodsfor preparing administrable compositions will be known or apparent tothose skilled in the art and are described in more detail in suchpublications as Remington's Pharmaceutical Science, 15th ed., MackPublishing Company, Easton, Pa. (1980).

Dosages for typical therapeutics, particularly for PPIs, are well knownto those of skill in the art. Moreover, such dosages are typicallyadvisorial in nature and may be adjusted depending on the particulartherapeutic context, patient tolerance, etc. Single or multipleadministrations of the compositions may be administered depending on thedosage and frequency as required and tolerated by the patient.

In preferred embodiments, the pentagastrin and PPI will be administeredin an amount sufficient to effect a measurable decrease in gastric acidsecretion, more preferably in an amount sufficient to effect asignificant decrease in gastric acid secretion (e.g., a statisticallysignificant decrease at the 90%, more preferably at the 95%, and mostpreferably at the 98% or 99% confidence level). The pentagastrin dosagewill range from about 0.05 to about 0.05 to about 25 μg/kg/hr,preferably from about 0.1 μg/kg/hr to about 15 μg/kg/hr and mostpreferably from about 0.5 μg/kg/hr to about 10 μg/kg/hr, while the PPIdosage, in preferred embodiments, will be consistent with currentclinical practice.

Similarly, where the pentagastrin and PPI are administered incombination with an antibiotic, the antibiotic is typically administeredin a manner and concentration consisting with clinical practice.

Uses of PPI and Pentagastrin Combinations.

The proton pump inhibitors are, as already mentioned, useful forinhibiting gastric acid secretion in mammals and man. In a more generalsense, they may be used for prevention and treatment of gastric-acidrelated diseases in mammals and man, including e.g. reflux esophagitis,gastritis, duodenitis, gastric ulcer and duodenal ulcer. Furthermore,they may be used for treatment of other gastrointestinal disorders wheregastric acid inhibitory effect is desirable, e.g. in patients on NSAIDtherapy, in patients with Non Ulcer Dyspepsia, in patients withsymptomatic gastro-esophageal reflux disease, and in patients withgastrinomas. They may also be used in patients in intensive caresituations, in patients with acute upper gastrointestinal bleeding, pre-and postoperatively to prevent aspiration of gastric acid and to preventand treat stress ulceration. Further, they may be useful in thetreatment of Helicobacter infections and diseases related to these.Other conditions well suited for treatment according to the methods ofthis invention include, but are not limited to Zollinger-Ellisonsyndrome (ZES), Werner's syndrome, and systemic mastocytosis.

The methods and formulations of this invention are suitable for use inessentially any mammal and this invention embraces veterinary as well ashuman medical applications. Thus, the methods of this invention areapplicable to humans and non-human mammals (e.g. a rodent, largomorph,bovine, canine, equine, non-human primate, etc.).

Therapeutic Kits.

In another embodiment, this invention provides therapeutic kits forpractice of the methods of this invention. Such kits preferably includea container containing one or more proton pump inhibitor(s) and acontainer containing pentagastrin and/or a pentagastrin/gastrin analogueor derivative. Both the pentagastrin and the PPI(s) can be in onecontainer or they can be in separate containers. In certain embodiments,the “pentagastrin/gastrin/analogue” and/or the PPIs are provided in adry form, while in other embodiments, the“pentagastrin/gastrin/analogue” and/or PPIs are suspended, or dissolvedin an excipient/buffer.

In certain embodiments, the kits optionally include one or moreantibiotics, e.g. an antibiotic selected from the group consisting ofpenicillin based antibiotics, tetracyclines, macrolides, cephalosporins,and fluoroguinolones.

The kit can comprise packaging that retains and presents the medicantsat separate respective consecutive locations identified by visiblydiscernible indicia and the times at which the medicants are to be takenby the patient. In various embodiments, the times can include each dayof the week and specified times within each day presented in the form ofa chart located on one face of the package wherein the days of the weekare presented and the times within each day the medicants are to betaken are presented in systematic fashion.

In addition, the kits can include instructional materials containingdirections teaching the use of a pentagastrin, a gastrin, or aderivative or analogure thereof in combination with one or more PPIs toenhance the efficacy of the PPI. While the instructional materialstypically comprise written or printed materials they are not limited tosuch. Any medium capable of storing such instructions and communicatingthem to an end user is contemplated by this invention. Such mediainclude, but are not limited to electronic storage media (e.g., magneticdiscs, tapes, cartridges, chips), optical media (e.g., CD ROM), and thelike. Such media may include addresses to internet sites that providesuch instructional materials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Inhibition of Pentagastrin-Induced Gastric Acid Secretion byIntravenous Pantoprazole: A Dose-Response Study

The purpose of this study was to compare the gastric acid inhibitoryability of increasing doses of intravenous (i.v.) pantoprazole with thatof i.v. famotidine and placebo. Pentagastrin was infused continuously inhealthy subjects as a model for patients with Zollinger-Ellisonsyndrome.

In brief, pentagastrin (1 μg/kg/h) was infused to stimulate maximum acidoutput in 39 subjects over a 25-h period. After 60 min of pentagastrininfusion, subjects received a single dose of i.v. pantoprazole (20, 40,80, or 120 mg), i.v. famotidine (20 mg), or saline placebo. Thevariables measured were onset of response (time until acid output fellto <10 mEq/h), duration of response (time acid output remained <10mEq/h), and cumulative acid output over 24 h.

All doses of i.v. pantoprazole produced a dose-dependent suppression ofacid output to <10 mEq/h. Single i.v. doses of pantoprazole, 80 and 120mg, suppressed acid output by >90% in all subjects for <21 h and had anonset of action of <1 h. Intravenous pantoprazole had a rapid onset anda clear dose-related effect, with a significantly longer duration ofaction than that of i.v. famotidine.

Introduction.

Zollinger/Ellison syndrome (ZES), a gastric hypersecretory conditionthat results from high circulating levels of gastrin, often requireshigh and repeated doses of acid output (AO) inhibiting agents to reduceeffectively intragastric acidity. Although histamine H2-receptorantagonists have been used successfully to treat ZES in the past, theerratic and diminishing responses with theses antagonists (McCarthy andHyman (1982) Dig. Dis. Sci., 27: 353-357), as well the progressiveoccurrence of more severe side effects associated with the use of largerdoses (Drogen et al. (1978) Drugs, 15: 93-131), has led to the use ofmore effective proton pump inhibitors (PPIs).

Parenteral antisecretory drugs are often required in patients with ZESwho have nausea, vomiting, or severe diarrhea (Von Schrenck et al.(1988) Gastroenterology, 1326-1334; Fox et al. (1974) Surg. Clin. North.Am., 54: 395-407). This occurs during the preoperative period, duringthe administration of chemotherapy and in the setting of uppergastrointestinal bleeding. However, currently there are no intravenous(i.v.) dosage forms of PPIs approved for use in the United States.Although effective in high doses, i.v. H2-receptor antagonists must becontinuously infused, which is not always possible in ZES patients(Saced et al. (1989) Gastroenterology, 96: 1393-1402; Fraker et al.(Surgery, 104: 1054-1063).

Pantoprazole, the newest PPI, has been shown to be as potent whenadministered by the i.v. route as by the oral route in short termstudies (Hartmann and Ehrlich (1998) Aliment. Pharmacol. Ther., 12:1027-1032; Simon et al. (1990) Aliment Pharmacol. Therap., 4: 239-245).However, no i.v. dose-response studies have previously been performedduring the continuous administration of i.v. pentagastrin over a 24-hperiod. We attempted to extend the knowledge obtained from a previousstudy (Simon et al. (1990) Z. Gastroenterol., 28: 443-447) to a 24 h andto predict the minimum effective, single i.v. dose of pantoprazoleneeded to control AO in patients with ZES, by developing a continuouspentagastrin infusion model that would stimulate maximal gastric AO andthereby mimic ZES in healthy volunteers.

The intent of this study is to provide an informative pharmacologicalmodel for the management of ZES by predicting the minimum effective doesof i.v. pantoprazole needed to control AO, and to compare the efficacyand duration of various single doses of i.v. pantoprazole with those ofsingle doses of i.v. famotidine (20 mg) and placebo to suppresspentagastrin (PG)-stimulated AO over a 24-h period.

Materials and Methods.

A total of 39 healthy men and women, 18-45 yr of age (mean age, 31.4±6.7yr:male/female ratio, 2:1), were selected for this open label, singledose, single site, parallel treatment study. The study protocol wasapproved by the UCLA Institutional Review Board and conducted accordingto the provisions of the Declaration of Helsinki and its amendments.Written informed consent was obtained from each participant beforeenrollment, and the identity of each patient was kept confidential.

Each subject underwent a prestudy screening that included a physicalexamination, electrocardiogram (ECG), laboratory blood and urineanalysis, and a pregnancy test for women. The laboratory test included acomplete blood count, blood chemistry tests, a test for humanimmunodeficiency virus, a screening for hepatitis B and C, and a urinedrug screen. Subjects were excluded from the study if abnormalities inthese parameters were detected. Maximum AO (MAO) were also determinedduring the screening period. MAO was estimated by the subcutaneousinjection of PG (6 μg/kg), after which four 15-min samples werecollected and analyzed (Table 1). Subjects who were huposecretors (MAO<5 mEq/h), who tested positive for Helicobacter pylori (H. pylori), orwho had a history of peptic ulcer disease were excluded from the study.Subjects who completed the screening examination and were eligible forthe study were assigned to one of six treatment groups to receive i.v.doses of pantoprazole (20, 40, 80, or 120 mg), i.v. famotidine (20 mg),or placebo (normal saline). Pantoprazole (manufactured by Byk Gulden andsupplied by Wyeth-Ayerst Research) in the form of lyophilized powder (40mg) was reconstituted with normal saline in a quantity sufficient tomake 90 ml.

To stimulate MAO, subjects received an infusion of PG for 25 h(Peptavalon, Wyeth-Ayerst; 1.0 μg/kg/h) begun 1 h before the start ofthe study drug infusion. Gastric aspirates were collected by means ofnasogastric (NG) tube in 15-min fractions from 1 h before study drugtreatment through the end of treatment h 2 and in 30 min fractions fromh 3 through the end of h 24. TABLE 1 Acid output values afterintravenous treatment. Screening Acid Output (mEq/h) Treatment PeriodTreatment Period (MAO) −1-0 h 0-6 h 6-12 h 12-18 h 18-24 h Pantoprazole(mg) 20 (n = 4-6) 36.6 ± 9.0 18.8 ± 8.8  11.7 ± 10.5 13.6 ± 13.3 15.1 ±13.7 14.2 ± 8.6  40 (n = 8) 40.1 ± 7.8 22.3 ± 13.0 8.1 ± 5.4 4.8 ± 4.07.3 ± 4.1 10.1 ± 3.7  80 (n = 8) 27.1 ± 4.5 18.8 ± 17.4 2.4 ± 1.5 1.2 ±0.6 3.7 ± 1.9 6.1 ± 3.2 120 (n = 4-5) 16.9 ± 3.6 6.9 ± 4.8 1.3 ± 0.8 0.7± 0.2 3.7 ± 0.7 5.2 ± 0.4 Famotidine (mg) 20 (n = 4) 28.1 ± 7.0 29.1 ±15.4 4.4 ± 2.5 17.7 ± 5.1  25.8 ± 9.3  25.0 ± 10.4 Placebo (n = 3-8)42.8 ± 5.6 35.2 ± 9.3  34.1 ± 8.7  35.8 ± 8.7  34.8 ± 2.2  33.1 ± 9.6 

The variables measured were onset of response (time until AO decreasedto <10 mEq/h and remained below this level for 1 h), duration ofresponse (time from onset until time when AO levels rose to >10 mEq/hand remained so for 1 h), and cumulative AO over 24 h. The method ofcalculating onset and duration are shown in FIG. 1. The mean percentinhibition of acid production at the study periods of 0-6 h, 6-12 h,12-18 h, and 18-24 h was calculated for each study group. The percentinhibition was determined by comparing each group's mean pretreatmentMAO to the mean AO during the above study drug periods by using thefollowing formula:% inhibition=11−average rate of MAO over time indicated/average AO inthe period before study drug administrational (100%)

On the evening before study day 1, participants were admitted to theUCLA Clinical Research Center. The following morning (study day 1),after nasogastric (NG) tube placement and the basal collection period,subjects were started on continuous i.v. PG administration and MAOanalyses were performed. At the end of this period a single dose ofpantoprazole, famotidine, or placebo was infused in the opposite armover 15 min. Because no fluid or food p.o. was permitted during thestudy period, subjects received maintenance i.v. fluid to preventdehydration. Vital signs and electrolytes were monitored during thistime. On the morning of study day 2, at the end of the 25-h period,participants underwent a post study evaluation that included a physicalexamination, an ECG, and analyses of blood and urine.

The efficacy of the response to pantoprazole or famotidine wasdetermined by measuring levels of gastric acid secretion by usingstandard collection and assay techniques (White et al. (1973) Dig. Dis.,18: 7-13). Because this was an exploratory study, statistical analysisconsisted of presenting means and standard deviations.

Adverse events were monitored continuously in the Clinical ResearchCenter. Treatment-emergent adverse events were defined as adverse eventsnot present at baseline or events present at baseline that worsenedduring treatment.

Results

Model

During the screening period, a single subcutaneous injection of PGelevated acid output in all groups (Table 1). All MAO values appearednormal in this healthy population of volunteer subjects. The continuousinfusion of PG produced a significant and constant increases in AOduring the study. Mean cumulative AO increased in a linear fashion inthe placebo group over the 25-h period of PG stimulation (FIG. 2). Afteran initial burst of AO after the start of stimulation, AO fluctuatedbetween 20 and 50 mEq/h (mean, 34.5 mEq/h) during the course of thestudy (FIG. 3). Cumulative AO (0-24 h) reached a mean value of 829±86mEq. There was no evidence of tachyphylaxis to the gastricacid-stimulating effects of PG.

Onset

In general, the acid-suppressing activity of pantoprazole was observedwithin 15-20 min after administration. All doses of pantoprazolesuppresses AO to <10 mEq/h (FIG. 3). The onset of response (mean ±SD)was <1 h (0.8±0.3 h) for 80 mg pantoprazole, which was approximatelyequal to that seen with high dose i.v. famotidine (0.5±0.2 h) (FIG. 4A).Onset was faster with the 120-mg pantoprazole dose (0.4±0.2 h); however,the difference was not significant.

Duration

AO values (mean ±SD) at periods of 0-6 h, 6-12 h, 12-18 h, and 18-24 hafter i.v. treatment are shown in Table 1. Peak inhibition was reachedat approximately 3-43 h for all doses: 83% inhibition in the 20-mg dosegroup, 90% inhibition in the 40-mg dose group, 99% inhibition in the80-mg dose group, and 100% inhibition in the 120-mg dose group. Peakinhibition was 94% for famotidine. The duration of action for 80 mg ofpantoprazole was markedly longer (21.2±1.9 h) than with 20 mg (8.4±8.0h), 40 mg (15.9±7.6 h) of pantoprazole, or 20 mg (6.5±2.1 h) offamotidine (FIG. 4B). Although 40 mg pantoprazole suppresses AO forapproximately 16 h, the time to onset and duration of action were morevariable among individual subjects in this dose group compared withthose subjects given 80 and 120 mg of pantoprazole. The doses ofpantoprazole with >90% inhibition (i.e., 40, 80, 120 mg) had no longerdurations of action than did 20 mg of famotidine.

Average AO Over 24-h Period

After 1 h, both the 80 and the 120-mg doses of pantoprazole suppressedAO to <10 mEq/h in all subjects, where it remained for the full 24-hperiod (FIG. 2), whereas famotidine lost effectiveness after 6 h.Intravenous pantoprazole reduced cumulative AO in a dose-dependentmanner. When averaged over the 24-h period, a dose of 20 mg and 40 mgpantoprazole reduced the hourly AO to 11.9 mEq/h and 7.6 mEq/hrespectively. The lowest average rate over the 24 h was seen in the 120mg group (2.4 mEq/h), followed closely by 80 mg pantoprazole (3.3mEq/h). Famotidine had an average ratio over the 24-h period of 18.2mEq/h.

Safety

Of the 39 subjects enrolled, 35 completed the study. There were noserious adverse events observed. The most frequently reported adverseevents were dyspepsia, which was mild in most cases, and mild gastricbleeding. Four subjects withdrew from the study because of there adverseevents. Three subjects receiving i.v. pantoprazole withdrew from thestudy because of increasing dyspepsia or nausea, and one receivingplacebo withdrew because of dyspepsia and bleeding. No subjects from thefamotidine group withdrew because of adverse events. Three subjects fromthe famotidine group complained of abdominal pain and dyspepsia and werenoted to have small amounts of blood in their NG aspirate. None of theadverse events were judged by the investigator to be related to any ofthe study drugs and could have been due to the administration of i.v.pentagastrin. All subjects' symptoms disappeared shortly afterdiscontinuations of the study. No clinically important abnormallaboratory results or other findings, including ECGs, were reported.None of the subjects reported visual changes.

Discussion.

The results of this study demonstrated that single doses of i.v.pantoprazole ranging from 20 mg to 120 mg suppressed gastric acidsecretion, in a dose-dependent manner, to <10 mEq/h in healthyvolunteers subjects to continuous PG-induced hypersecretion. The dosesof pantoprazole producing >90% inhibition (i.e., 40, 80, and 120 mg) hada longer duration of action than did famotidine. Although famotidineeffectively reduced AO, it had a relatively short duration of actioncompared with that of pantoprazole. Pantoprazole doses of 80 mg and 120mg had an onset of action <1 h and suppresses acid output (99-100%) to<10 mEq/h for >20 h. No difference in cumulative gastric acid output wasobserved between the group receiving 80 mg and that receiving 120 mg ofpantoprazole. These data suggest that a single dose of 80 mg i.v.pantoprazole will rapidly and effectively control acid secretion in mostpatients with gastric acid hypersecretory states, such as those who haveZES.

Because the clinical manifestations of ZES, including death, are almostentirely due to the effects of hypergastrinemia (Fox et al. (1974) Surg.Clin. North. Am., 54: 395-407; Ellison and Wilson (1964) Ann. Surg.,160: 512-530; Jensen et al. (1983) Ann. Intern. Med., 989: 59-75), areduction of AO to normal levels, i.e., <10 mEq/h, has proved to be aneffective treatment and prolongs survival. In some situations, howeverit is advantageous to inhibit gastric acid secretion via the i.v. routeof administration: for example, in unconscious ZE patients, those withupper gastrointestinal bleeding, or in patients with metastatic diseasereceiving chemotherapy (Von Schrenck et al. (1988) Gastroenterology,1326-1334). Thus, there is an urgent need for a parenteral antisecretorydrug that can reduce AO, safely and effectively, to clinically tolerablelevels. Currently there are no i.v. forms of PPIs approved for use inthe United States. Although the histamine H2 receptors antagonistscimetidine (Ronfils et al. (1979) World J. Surg., 3: 597-604; McCarthy(1978) Gastroenterology, 74: 453-458), ranitidine (Howard et al. (1985)Gastroenterology, 88: 1026-1033), and famotidine (Howard et al. (1985)Gastroenterology, 88: 1026-1033; Campoli-Richards (1986) Drugs, 32:197-221) effectively and safely control gastric acid hypersecretion inpatients with ZES, they all have certain disadvantages; these includethe need for high and more frequent dosing (Rume et al. (1981)Gastroenterology, 80: 1265), the occurrence of tachyphylaxis (Devency etal. (1983) Am. J. Surg., 146: 116-123), the need for continuous i.v.infusion (Saced et al. (1989) Gastroenterology, 96: 1393-1402; Fraker etal. (Surgery, 104: 1054-1063), and lack of response in some patients(Drogen et al. (1978) Drugs, 15: 93-131; Vallot et al. (1983) Dig. Dis.Sci., 28: 577-584). The rapid loss of antisecretory efficacy with theseagents may account for the failure in the treatment of patients with ZESsyndrome, in which high doses of H2-recepor antagonists must be used. Inour study, all doses of pantoprazole had a longer duration of actionthan did famotidine and, thus, would appear to be preferable to i.v.famotidine for controlling gastric acid secretion in patients with ZES.

In addition, our investigation showed that i.v. PG (1 pg/kg/h)administered for ≦25 h created a continuous hypersecreory acidicenvironment with no indication of tachyphylaxis, as evidenced by theincrease in gastric acid secretion at the end of the 24-h period for allsubjects in all treatment groups, including the placebo group. Thisstudy was one of the first to use PG for a 25-h duration and extends thetime length of a previous 4-h study (Simon et al. (1990) Z.Gastroenterol., 28: 443-447). Results using healthy subjects have shownthat 80% maximal gastric secretory response is reached in approximately80% of subjects given a PG dose of 0.6 μg/kg/h (Mason et al. (1969) Gut,10: 34-38; Wormsley et al. (1966) Lancet, 1: 993-996). The dose of 1pg/kg/h was chosen because it is tolerable and produces a near maximalacid secretion that is sustainable for 24 h in healthy subjects and thatis comparable to the lower range of AO seen in patients with ZES (Masonet al. (1969) Gut, 10: 34-38; Wormsley et al. (1966) Lancet, 1: 993-996;Chin et al. (1986) J. Clin. Pharmacol., 26: 281-285). This PGstimulation model has been shown to be particularly relevant to thestudy of the Zollinger-Ellison syndrome (Hirschowitz et al. (1995) Dig.Dis. Sci., 40(suppl): 3S-23S).

There were no serious adverse events in this study. The most frequentlyreported event was mild to moderate dyspepsia. This was experienced bysubjects who were administered the pantoprazole 20-mg dose, i.v.famotidine, or placebo, and was probably due to the unopposed effects ofgastric acid secretion, or to PG or NG tube protocol procedures. A smallnumber of subjects were noted to have modest amounts of blood in theirNG aspirate; however, this was believed to be related to NG tube traumaor to the effects of continuous pentagastrin.

In conclusion, these results indicate that a single dose of i.v.pantoprazole rapidly and effectively reduces gastric acid secretion in adose-dependent manner to normal levels in subjects exposed to continuousi.v. PG infusion. A single dose of i.v. pantoprazole, either 80 mg or120 mg, reduced AO levels as quickly and more potently than did i.v.famotidine 20 mg, and had a duration of action approximating 24 h.Furthermore, these data predict that gastric acid secretion can bysafely and effectively controlled by i.v. pantoprazole at a dose of 80mg in patients with gastric acid hypersecretory disorders such as ZES,and that i.v. pantoprazole is preferable to i.v. famotidine. Although,in this study, i.v. pantoprazole rapidly and effectively inhibitedgastric acid secretion in subjects following stimulated conditions suchas with i.v. pentagastrin, its efficacy in unstimulated conditions suchas in intensive care settings was not assessed here, but deservesfurther investigation.

Example 2 Discovery of Gastrin Receptors in the Kidney Reveals HormonalCoupling of Digestion and Excretion

Ingestion of a meal results in acute changes in renal function necessaryto handle absorbed nutrients (Pullman (1954) J. Lab. Clin. Med. 44:320-332; Jolliffe and Smith (1931) Am. J. Physiol. 99: 101-107;Schoolwerth et al. (1975) Kidney Int. 7: 397-404). However, themechanism for communication between the digestive and renal systems hasnever been established. In this example, we report that receptors forgastrin, cholecystokinin type B/gastrin receptors (CCKB/gastrin), areexpressed at high levels in the kidney and that gastrin, elevated byeither a meal or direct renal infusion, stimulates an increase inurinary Na⁺ excretion and urine volume that is inhibited by theCCKB/gastrin receptor-specific antagonist, L-365,260 (Lotti and Chang(1989) Eur. J. Pharmacol. 163: 273-279). Gastrin stimulated excretion ofsodium and water and its reversal by L-365,260 are paralleled byCCKBR/gastrin receptor mediated inhibition and reversal of inhibition ofrenal tubular Na⁺-K⁺-ATPase activity, respectively. The identificationand functional characterization of renal tubular gastrin receptorsreveals the direct link between digestion and renal excretion mediatedby the principle meal-stimulated gastrointestinal hormone, gastrin.

Introduction:

Nutrients absorbed during a meal present an acute load to the kidneysthat are responsible for maintaining tight control of metabolitesincluding water and electrolytes. It has been hypothesized for manyyears that gastrointestinal hormones that become elevated in response toa meal may play a role in the acute regulation of the renal handling ofabsorbed nutrient (Reinhardt et al.: (1975) Pflugers Arch. 354: 287-297;DeSanto et al. (1992) Ren. Physiol. Biochem. 51: 53-56; Bosch et al.(1983) Am. J. Med. 77: 873-879; Hostetter (1986) Am. J. Physiol. 250:F613-F618). Although several gastrointestinal hormones have been shownto indirectly influence renal function at pharmacologic concentrations,the actual meal stimulated hormones that are physiologicallyresponsible, as well as their site and mechanism of action, haveremained elusive. In humans, renal responses to protein meal ingestionled to a significant increase in the glomerular filtration rate (GFR),renal plasma flow (RPF) and an increase in salt excretion suggestingthat these effects were related to the ingestion of the protein meal(DeSanto et al. (1992) Ren. Physiol. Biochem. 51: 53-56; Hostetter(1986) Am. J. Physiol. 250: F613-F618). We hypothesized that gastrin,released following protein meals, may account for the renal responsesobserved in these investigations.

Previous studies have identified that the kidney is a major site forgastrin excretion that is not dependant on degradation of the peptide(Davidson et al. (1973) Gastroenterology 64: 955-961). A positivecorrelation between the renal functional mass and the ability of thekidney to extract gastrin support the role of the kidney as an organwith an important role in gastrin clearance (Kes et al. (1993) RenalPhysiol. Biochem. 16: 268-275). This role of the kidney in the abilityto clear gastrin has been best exemplified by the observation of anincrease in serum gastrin following nephrectomy in the rat (El Munshidet al. (1980) J. Physiol., 299: 157-171). Similarly, in humans there isa significant increase in the serum levels of gastrin once the serumcreatinine is elevated >3.0 mg/dl (Hansky (1979) World J. Surg. 3:463-467) that is reversed following renal transplantation (Nieksen etal. (1980) Acta Med. Scand. 207: 85-87).

The primary role of gastrin is believed to be in the regulation ofgastric acid secretion. Gastrin is released by gastric antral andintestinal G-cells into the circulation following a meal and remainselevated for up to two hours. Previous studies have identified thekidney as a major site for gastrin excretion that is not dependant ondegradation of the peptide (Schjonsby and Willassen (1977) Scand. J.Gastroenterol., 12: 205-207; Davidson et al. (1973) Gastroenterology 64:955-961). Following the cloning of the CCKB/gastrin receptor (Wank etal. (1992) Proc. Natl. Acad. Sci., USA, 89: 8691-8695), receptor tissuedistribution studies unexpectedly revealed a high level of both receptormRNA and protein in the kidney. This finding immediately suggested aphysiologic role for meal stimulated gastrin in the regulation of renalexcretion. The CCKB/gastrin receptor is a heptahelical, guaninenucleotide binding regulatory protein-coupled receptor known to couplevia Gαq to the activation of phospholipase C with the subsequentformation of inositol phospholipids and the release of intracellularcalcium (Wank et al. (1992) Proc. Natl. Acad. Sci., USA, 89: 8691-8695).To date, there have been no studies linking the CCKB/gastrin receptor tothe activation of Na+/K+ ATPase.

In this example, we demonstrate that CCKB/gastrin receptors are highlyexpressed in the kidney in an anatomic distribution functionallyconsistent with the actions of gastrin on the kidney. Physiologicconcentrations of gastrin either infused or naturally elevated inresponse to a meal act specifically at CCKB/gastrin receptors on renaltubular cells to increase the excretion of salt and water into theurine. Specific antagonism of meal stimulated gastrin indicates thatgastrin is the predominant mediator effecting meal induced renalexcretion. The observed gastrin related inhibition of renal tubularNa⁺-K⁺-ATPase activity may account for the mechanism of its actions. Thediscovery and functional characterization of renal tubular CCKB/gastrinreceptors strongly suggests that gastrin is the principle mediatorcoupling digestion and renal excretion.

Materials and Methods

Immunohistochemistry.

Rat kidney sections were cut, fixed in 4% formaldehyde in PBS for 6hours, dehydrated and paraffin embedded. Tissue blocks were cut into 4mm sections, deparaffinized, rehydrated and treated with 3% hydrogenperoxide for 5 minutes. After blocking with 20% swine serum, sectionswere incubated with rabbit anti-CCKB receptor antibodies directedagainst the third extracellular loop of the receptor (Tarasova et al.(1994) Letters in Peptide Science 1: 221-228) at 1:500 dilution followedby staining with LSAB-2 kit (DAKO Corp, Carpinteria, Calif.). The slideswere counter stained with Mayer's hematoxylin and observed under a NikonOptiphot II light microscope. Original magnification: A, 8×; B,C,F,100×; E, 400×.

Reverse Transcriptase-Polymerase Chain Reaction on Isolated RenalTubules.

Sections of kidney were digested with collagenase and microdissected toisolate individual glomeruli and tubular segments. RT-PCR (BoehringerMannheim) was performed on 1-4 mm segments of individual tubules.Primers corresponding to unique regions of the CCKBR that spanned anexon-intron junction were used to amplify target cDNAs from theindicated nephron regions under the following conditions (denaturationat 94° C. for 45 sec; annealing at 60° C. for 25 sec; extension at 72°C. for 60 sec for a total of 28 cycles with the final extension at 72°C. for 15 min.).

Renal Tubular Physiologic Responses.

Male Wistar-Kyoto rats (300 grams, 10-12 weeks old) (Harlan SpragueDawley, Indianapolis, Ind.) were maintained on a regular Purina rat chowdiet. Catheters were placed into the external jugular and femoral veinsand left carotid artery and systemic arterial pressure was monitored.The right suprarenal artery was cannulated with a PE-10 catheter heatstretched to 180 mm and a flow catheter was secured around the rightrenal artery (Transonic Systems, Ithaca, N.Y.). GFR was determined afteran intravenous infusion of normal saline containing [¹⁴C]-insulin (0.01mCi/10 ml (New England Nuclear, Boston, Mass.) at a rate of 5 ml/100 gbody wt. for 30 minutes followed by an infusion of 1.8 ml/100 g bodywt/hr). After an equilibration period of 120 minutes, 20 minutecollections were begun. Meal gavage studies were performed with 2.5ml/hr of liquefied rat chow administered via gastric tube. Gastrin 17-1(Peninsula Laboratories, Calif.) was diluted in normal saline andinfused (10 pmoles/kg/hr) via the right renal artery catheter. Followinga basal period, rats received either L-365,260 (80 pmoles/kg, 5 minbolus) or vehicle alone and 20 minute collection periods were begun fordetermination of sodium and water excretion.

Na⁺-K⁺-ATPase Activity.

Renal tubules (cortical and medullary thick ascending limb of Henle)were isolated (Slobodyansky et al. (1995) Am. J. Physiol. 268:F279-F284) and immediately homogenized (in a Polytron) in 4° C. reactionbuffer [4 mM MgCl₂, 100 mM tris(hydroxymethyl)aminomethane HCl, pH7.2].The dephosphorylation oftris(hydroxymethyl)aminomethane-p-nitrophenyl-phosphate was used as theindex of Na⁺-K⁺-ATPase activity (Eisner et al. (1997) Am. J. Physiol.273: R317-R323).

Results and Discussion

To determine whether CCKB/gastrin receptors are functionally positionedto mediate the proposed regulatory action of gastrin on renal excretion,the cellular localization of CCKB/gastrin receptors within the kidneywas determined by immunohistochemistry. Specific antibodies directedagainst the third extracellular loop of the CCKB/gastrin receptor (12)were used for staining rat, guinea pig and human kidney tissue sections.Similar to guinea pig and human, immunostaining of CCKBR expression inrat kidney was moderate in the cortex (CT), most intense in the innerstripe of the outer medulla (OMIS), less intense in the outer stripe ofthe outer medulla (OMOS) and absent from the inner medulla (IM). Highermagnification revealed cortical CCKBR expression in proximal tubules(PT), particularly in early segments found to be in continuity withBowman's capsule of the glomerulus (GLOM)). A higher magnification ofthe junction between the inner stripe of the outer medulla (OMIS) andthe inner medulla (IM) revealed the striking localization of staininglimited to the inner stripe that was also present at the basolateralsurface of cells within the medullary thick ascending limb (MTAL).Occasional CCKB/gastrin receptor positive cells within the glomerulus(GLOM) appeared to be either on the outer aspect of the glomerular tuftconsistent with visceral epithelial cells (single arrow) or less wellidentified cells located deeper within the tuft (double arrow).Immunohistochemistry also localized CCKB/gastrin receptor on afferentarterioles and medium sized blood vessels within the kidney. Corticaltissue processed with pre-immune serum showed only trace staining ofvascular lumina consistent with non-specific erythrocyte peroxidaseactivity. Overall, the relative level of CCKB/gastrin receptorexpression detected by immunohistochemistry in the kidney wassignificantly higher than in the stomach, an organ well characterizedfor CCKB/gastrin receptor expression.

RT-PCR using oligonucleotide primers based upon the rat CCKB/gastrinreceptor cDNA sequence amplified a product of the expected size (344 bp)(Wank et al. (1992) Proc. Natl. Acad. Sci., USA, 89: 8691-8695) fromtotal RNA isolated from microdissected, isolated segments of nephrons.Consistent with their localization by immunohistochemistry, RT-PCRdetected transcripts in the glomerulus, proximal tubule, and collectingduct. No PCR products were obtained from the distal convoluted tubule.

The physiologic regulation of renal function through the CCKB/gastrinreceptor was investigated in male Wistar-Kyoto rats. Renal hemodynamicand tubular functions were measured following meal ingestion by gastricgavage with liquefied rat chow (previously shown to increase serumgastrin two-fold (Aurang et al. (1997) Am. J. Physiol. 272:G1243-G1248)) either alone or with the addition of a maximal inhibitorydose of the CCKB/gastrin receptor specific antagonist, L-365,260 (FIGS.5A and 5B). Meal ingestion caused a small but significant increase inglomerular filtration rate (GFR) and renal blood flow (RBF) compared tobasal values that was inhibited by L-365,260 administration (FIG. 3,panels a and b). In parallel with the meal stimulated elevation in serumgastrin (data not shown), urinary sodium (UNaV) and fractional excretionof sodium (FeNa=UNa⁺×V/GFR [SNa⁺]), a measure of the renal tubularreabsorption of sodium, both increased more than 5-fold. The increasesin UNaV and FeNa were both suppressed to 2-fold in the presence ofL-365,260 (FIG. 5C and FIG. 5D). In parallel with the increase in sodiumexcretion during the meal, the urinary flow rate more than doubled (FIG.5E). This meal-stimulated increase in urine flow rate was nearlycompletely inhibited in the presence of L-365,260 compared to vehiclealone (FIG. 5E). Gastric gavage with an equivalent volume and sodiumconcentration as the liquified rat chow (used as a negative control) hadno significant effect on the above parameters of renal function duringthe study period (data not shown). The ability of L-365,260 to inhibitup to 80% of the meal-stimulated increase in sodium and water excretionindicates that gastrin is responsible for the majority of this effectand that other meal stimulated hormones have a minor role in sodiumregulation in response to a meal. Because the proximal tubule is themajor site of solute and water reabsorbtion, the most likely site ofaction of postprandially released gastrin is at CCKBR expressed on theproximal tubules. The observation that gastrin increases the fractionalsodium excretion to a greater extent than renal blood flow also providesevidence that the site of gastrin action in the kidney is at the levelof the proximal tubule.

To exclude extrarenal actions of gastrin that may indirectly influencerenal function, gastrin was infused directly into the right renal arteryat previously determined postprandial concentrations and renalhemodynamic and tubular responses were compared to baseline and thecontrol infusion of vehicle alone in the left renal artery. Renalhemodynamic and tubular responses to infused gastrin were determinedseparately for the infused right kidney compared to the non-infused leftkidney. Gastrin administration caused small but significant increases inRBF (FIG. 6A) compared to basal values and GFR (FIG. 6B) in the rightkidney compared to the control left kidney (FIG. 6B). This is consistentwith the presence of CCKBRs identified by immunohistochemistry on renalarterioles as well as previous studies demonstrating peripheralvasodilatory effects of gastrin in rat (Guth (1991) Scand. J.Gastroenterol. 26 (suppl. 180): 118-125). Urinary sodium excretion(UNaV) and fractional sodium excretion (FeNa) were measured followinginfusion of gastrin into the right renal artery alone or with subsequentadministration of L-365,260. Simultaneous urine volumes were determinedby separate ureteral catheters. Urinary sodium excretion was increasedmore than four-fold by gastrin infusion to the right kidney compared tobaseline, the recovery period and the control left kidney (FIG. 6C).Following administration of L-365,260, the gastrin-stimulated elevationin the urinary excretion of sodium returned to near baseline valuesindicating that the results were specific for gastrin acting at theCCKBR. The fractional sodium excretion, FeNa, increased almost four-foldcompared to baseline (FIG. 6D). Similar to the results obtained for theurinary sodium excretion, following administration of L-365,260, FeNareturned to near baseline values. In parallel with the increase insodium excretion following gastrin administration, the urinary flow ratealmost doubled in the right kidney compared to the control left kidney(FIG. 6E). These changes in urine flow rate were completely antagonizedby L-365,260, and unaffected by vehicle alone, again indicating thespecific site of action at the CCKB/gastrin receptor. No significantchange in renal function was observed in the control left kidney and therenal preparation remained stable throughout the eight period durationof the study (data not shown).

To further demonstrate the direct intrarenal action of gastrin and todetermine the biochemical mechanism for gastrin's natriuretic activity,the effect of the physiological elevation of meal stimulated gastrin onNa⁺-K⁺-ATPase activity was measured in the proximal tubule (PT) andmedullary thick ascending limb of Henle (mTAL). Following a meal,Na⁺-K⁺-ATPase activity was significantly inhibited in both the PT(12.79±1.37%) and mTAL (13.58±1.09%) compared to unfed control rats(FIG. 7). The complete reversal of this meal induced inhibition ofNa⁺/K⁺-ATPase activity by direct intrarenal infusion of the CCKB/gastrinreceptor specific antagonist, L-365,260, prior to the meal indicatesthat the CCKB/gastrin receptor mediates the full inhibitory effect ofthe meal. (FIG. 7). The decrease in the Na⁺-K⁺-ATPase activity with ameal and it's reversal by the CCKBR specific antagonist were paralleledby an increase and reversal in sodium excretion and urine flow,respectively (data not shown).

Intestinal cholecystokinin (CCK), another potent agonist at theCCKB/gastrin receptor is also released in response to a meal. However,it is unlikely to account for the effect of a meal on renal functionbecause of the relatively low circulating level of CCK. Although thetype A cholecystokinin receptor is also expressed in the kidney (15),gastrin has extremely low affinity for this receptor.

The results reported here demonstrate that CCKB/gastrin receptors areexpressed at high levels within the renal tubule where they are directlystimulated by meal induced elevations in serum gastrin to increase theexcretion of sodium and water absorbed during the meal. The potentinhibitory effect of gastrin on Na⁺-K⁺-ATPase activity demonstrated inthis study is the likely intracellular mechanism responsible for theobserved decrease in Na⁺ reabsorbtion in the proximal tubule andmedullary thick ascending limb of Henle. However, other cellularmechanisms such as inhibition of the Na⁺/H⁺ exchanger (NHE3) or increasein tubular cGMP-mediated regulation of the cystic fibrosis transmembraneconductance regulator (CFTR) as suggested for uroguanylin may also beinvolved (Yun et al. (1997) Proc. Natl. Acad. Sci., USA, 94: 3010-3015;Forte et al. (1996) Am. J. Kid. Dis. 28: 296-304).

While this study does not exclude other neurohormonal factors, itstrongly supports gastrin as the major integrator between thegastrointestinal and renal systems following a meal. Other mealstimulated gastrointestinal hormones have been shown to affect renalfunction, such as glucagon and VIP, however, their effect on renalhemodynamics and tubular function occurs only at pharmacologicalconcentration (Briffeuil et al. (1996) J. Metabolism 45: 383-388; Nairet al. (1994) Diabetes Care 17: 711-716; Lonergan and Field (1991) Clin.Exp. Pharmacol. Physiol. 18: 819-826). A number of non-gastrointestinalhormones and peptides that are indirectly elevated following a meal,such as prostaglandins, adrenomedulin, atrial natriuretic peptide,kinins, and substance P, affect renal tubular function indirectly (Reddyet al. (1998) Nephron 79: 192-200; Jougasaki et al. (1997) Am. J.Physiol. 272(2): F260; Long et al. (1990) Prostaglandins 40: 591-595;Fildes and Atkins (1997) Am. J. Physiol. 272(5 Pt 2):R1396-1401; Capassoet al. (1989) Pflugers Arc. 415: 336). Therefore, gastrin is the firstmeal-stimulated gastrointestinal peptide hormone shown to effect anatriuresis at physiologic levels through a direct receptor mediatedaction on renal tubules. Recent data for pentagastrin stimulated urinarysodium excretion in human subjects (Example 1) indicate that thefindings presented in this study have important clinical implicationsregarding nutritional and pharmacological factors regulating the renalreabsorbtion of solutes and water and may ultimately lead to noveltherapeutic strategies for improved salt and water homeostasis.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A method of increasing the efficacy of a gastric H⁺/K⁺-ATPase pumpinhibitor (PPI) in a mammal, said method comprising: administering tosaid mammal a pentagastrin, a gastrin, or analogue thereof inconjunction with said gastric proton pump inhibitor.
 2. The method ofclaim 2, wherein said a pentagastrin, a gastrin, or analogue thereof ispentagastrin.
 3. The method of claim 2, wherein said mammal is a mammaldiagnosed with a pathology characterized by excess gastric acidsecretion.
 4. The method of claim 3, wherein said pathology is selectedfrom the group consisting of Zollinger/Ellison syndrome (ZES),gastroesophageal reflux disease (GERD), peptic ulcer disease, atrophicgastritis, esophagitis, and idiopathic gastric acid hypersecretion. 5.The method of claim 2, wherein said mammal is a human.
 6. The method ofclaim 2, wherein said administering comprises administering saidpentagastrin prior to administration of said gastric proton pumpinhibitor.
 7. The method of claim 2, wherein said administeringcomprises administering said pentagastrin simultaneously toadministration of said gastric proton pump inhibitor.
 8. The method ofclaim 2, wherein said proton pump inhibitor is selected from the groupconsisting of rabeprazole, omeprazole, lansoprazole, pantoprazole, andcogeners or racemic mixtures thereof.
 9. The method of claim 2, whereinsaid pentagastrin is administered by subcutaneous injection.
 10. Themethod of claim 2, wherein said pentagastrin is administered in a dosageranging from about 0.1 mg/kg/hr to about 10 mg/kg/hr.
 11. The method ofclaim 1, wherein said mammal is a human.
 12. The method of claim 1,wherein said mammal is a non-human mammal.
 13. A kit for the treatmentof a pathology characterized by excess gastric acid secretion, said kitcomprising: a container containing a proton pump inhibitor (PPI); and acontainer containing a pentagastrin, gastrin, or analogue thereof. 14.The kit of claim 24, wherein said a pentagastrin, gastrin, or analoguethereof is pentagastrin.
 15. The kit of claim 14, wherein said protonpump inhibitor is selected from the group consisting of rabeprazole,omeprazole, lansoprazole, pantoprazole, and or cogeners and racemicmixtures thereof.
 16. The kit of claim 14, wherein said PPI is presentin a pharmaceutically acceptable excipient or diluent.
 17. The kit ofclaim 14, wherein said PPI is dehydrated.
 18. The kit of claim 14,wherein said pentagastrin is present in a pharmaceutically acceptableexcipient or diluent.
 19. The kit of claim 14, wherein said pentagastrinis dehydrated.
 20. The kit of claim 14, further comprising anantibiotic.
 21. The kit of claim 20, wherein said antibiotic is selectedfrom the group consisting of penicillin based antibiotics,tetracyclines, macrolides, cephalosporins, and fluoroguinolones.
 22. Thekit of claim 14, wherein said kit further comprises instructionalmaterials describing the use of pentagastrin, gastrin, or an analoguethereof in conjunction with a PPI to reduce gastric acid secretion.